Explanation for the labeling of cervical motoneurons in young rats following the introduction of horseradish peroxidase into the calf.

This study was carried out to determine whether cervical motoneurons, labeled following the introduction of horseradish peroxidase into the rat hind leg, belong to the cutaneous trunci motoneuron pool. The cutaneous trunci is a superficial muscle that extends from the axilla, over the flank, and into the thigh. Its nerve supply is derived from the brachial plexus. In experimental animals, horseradish peroxidase was either injected directly into the right gastrocnemius muscles, or applied to gelfoam and implanted over the calf muscles in the right leg of 5-, 10-, 15-day-old and adult rats. In control animals the cutaneous trunci was denervated prior to the administration of horseradish peroxidase. Labeled cervical motoneurons were present in the 5-, 10-, and 15-day-old but not the adult experimental groups and were located within the predetermined confines of the cutaneous trunci motoneuron pool. No labeling of cervical motoneurons was observed in any of the control groups in which the cutaneous trunci muscle was denervated. The most likely explanation for the labeling of cervical motoneurons in young rats was the local diffusion of horseradish peroxidase from the calf to the thigh, where it entered the cutaneous trunci muscle and was taken up by some of its motoneurons. The absence of such labeling in adult rats was probably due to the presence of connective tissue barriers to diffusion and to the greater distance between the site of horseradish peroxidase application and the cutaneous trunci muscle, which prevented the tracer from reaching the cutaneous trunci muscle and labeling its motoneurons.

Proteins of slow axonal transport in sciatic motoneurones of rats with streptozotocin-induced diabetes or galactosaemia.

This study examined the distribution of axonally transported tubulin and a 68 kDa polypeptide in the sciatic nerve 34 days after injection of labelled methionine into the ventral horn of the spinal cord of control rats, rats with streptozotocin-induced diabetes mellitus and rats fed a diet containing 40% galactose. The proteins were separated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) of pellets produced by treatment of nerve extracts with Triton X-100 followed by differential ultra-centrifugation. The most marked effect of both diabetes and galactosaemia was to reduce the amount of activity present in tubulin transported at a rate of 1.4 to 2.1 mm/day. The distribution of activity in the 68 kDa polypeptide band was not markedly affected by either of the experimental conditions. These findings, taken together with those of other studies, indicate that the polyol pathway may contribute to the development of some defects of nerve function in diabetic rats, but is uninvolved in others.

Alterations in levels of mRNAs coding for neurofilament protein subunits during regeneration.

Animal models of neuronal injury can be used to explore mechanisms that regulate the expression of genes coding for cytoskeletal proteins and transmitter-related markers. In the present study, in situ hybridization was used to measure levels of messenger ribonucleic acid (mRNA) encoding each of the neurofilament subunits and beta-tubulin in spinal motor neurons at intervals (4 to 56 days) following a unilateral crush of the sciatic nerve. Levels of beta-tubulin mRNA increased (approximately twofold), peaked at 28 days postaxotomy, and returned to control values by 56 days postaxotomy. In contrast, levels of mRNA encoding neurofilament subunits were reduced and returned to control values at 56 days following the lesion. There were significant differences among relative levels of mRNAs coding for each subunit. Other studies have demonstrated that the ratio of pulse-labeled neurofilament subunits in motor axons remained unaltered during regeneration. Therefore, the ratios of neurofilament subunits in axons must be regulated at one of the steps that intervenes between the control of levels of mRNA and the anterograde axonal transport of assembled neurofilaments.

Agrin-like molecules in motor neurons.

According to the agrin hypothesis molecules that mediate the nerve-induced aggregation of acetylcholine receptors and acetylcholinesterase on developing and regenerating skeletal muscle fibers are similar or identical to agrin, a protein extracted from the electric organ of marine rays. Here we present evidence that agrin is highly concentrated in the cell bodies of motor neurons and is transported to axon terminals which is consistent with the agrin hypothesis.

Immunocytochemical study of the relations of acetylcholinesterase, enkephalin-, substance P-, choline acetyltransferase- and calcitonin gene-related peptide-immunoreactive structures in the ventral horn of rat spinal cord.

Calcitonin gene-related peptide (CGRP)-like immunoreactivity was localized immunocytochemically in the large motoneurons in the ventral horn of rat spinal cord. Using fluorescence double-labelling substance P (SP)-immunoreactive nerve fibres were found to surround both the CGRP-positive and negative motoneurons, whereas enkephalin (ENK)-immunoreactive fibres surrounded mainly CGRP-negative cells. All CGRP-like immunoreactive motoneurons were also choline acetyltransferase (ChAT)- and acetylcholinesterase (AChE)-positive. On the other hand a large population of ChAT- and AChE-positive motoneurons were devoid of CGRP-immunoreactivity. It is probable that CGRP/ChAT/AChE-positive cells surrounded by SP-positive fibres have different functions in motoric nervous system than the CGRP-negative ChAT/AChE-positive cells, which are surrounded by ENK-immunoreactive fibres.

Ultrastructural localization of thyrotropin-releasing hormone immunoreactivity in the dorsal vagal complex in rat.

Thyrotropin-releasing hormone-like immunoreactivity (TRH-LI) was localized at the ultrastructural level in the dorsal vagal complex (DVC: dorsal motor nucleus of the vagus (DMV) and the nucleus of the solitary tract (NST] in rat. TRH-LI was concentrated in large granular vesicles in axons, presynaptic terminals, and non-synaptic axon varicosities. TRH-LI presynaptic terminals established both asymmetric and symmetric synaptic contacts with dendrites. These observations are consistent with recently described direct inhibitory and facilitatory effects of TRH on the electrical activity of neurons in the DVC.

Reorganization of intrinsic components in the distal motor axon during outgrowth.

We have studied the changes in the distribution of three intrinsic axonal components during the growth and maturation of sprouts in vivo. Neurofilaments, tubulin and synaptophysin, a synaptic vesicle protein, were visualized in motor axons and their sprouts using immunocytochemical staining of frozen longitudinal sections of muscle. We examined changes in these elements in sprouts regenerating after axonal crush injury and in those evoked from intact axons by denervation changes in muscle. Our results show that intrinsic axonal components move into newly formed motor axon sprouts in different temporal patterns. Based on the patterns of reorganization of staining of intrinsic axonal components, two types of outgrowth can be distinguished. One type, synaptic elaboration, is manifest by short, broad axonal processes that produce enlargement of the synaptic zone (synaptophysin staining) with little change in the distribution of intrinsic cytoskeletal elements. A second type of outgrowth, axonal elongation, occurs during axonal regeneration and ultraterminal sprouting and is longitudinal in form. In these sprouts there is a sequential appearance of neurofilament and then, several days later, tubulin immunostaining. Synaptophysin only accumulates in these sprouts after two weeks at points of synaptic contact with a muscle fibre.

Location of forelimb motoneurons in the Japanese toad (Bufo japonicus): a horseradish peroxidase study.

To label the spinal motoneurons innervating the forelimb muscles of the Japanese toad, horseradish peroxidase (HRP) was injected into these muscles or applied to the cut end of the brachial nerves (N. radialis and N. ulnaris). Spatial distribution of the HRP-labeled motoneurons was reconstructed from serial frontal sections of the spinal cord and their location was examined. Motoneurons innervating forelimb muscles were distributed in the lateral cell column from segment 3 to segment 5 of the ipsilateral brachial spinal cord. In the transverse plane of the spinal cord, motoneurons innervating the medial forearm muscles (innervated by N. ulnaris) were located in the more medial part of the lateral cell column, whereas those innervating the lateral forearm muscles and the upper arm muscle (innervated by N. radialis) were located in the more lateral part of the lateral cell column. Along the longitudinal axis of the spinal cord, motoneurons innervating the more anterior (flexor side) forearm muscles were located in the more rostral part of the spinal cord, whereas those innervating the more posterior (extensor side) forearm muscles were located in the more caudal part of the spinal cord. Thus, motoneurons innervating forearm muscles were well organized somatotopically not only in the transverse plane, but also along the longitudinal axis of the spinal cord. Such a somatotopic organization of motoneurons along the longitudinal axis could also be regarded as a functional one; the flexor motoneurons were located rostrally to the extensor motoneurons.

Pro-opiomelanocortin-derived peptides in transected and contralateral motor nerves of the rat.

An immunocytochemical method was used to reveal beta-endorphin and alpha-melanotropin peptides in nerve profiles at the neuromuscular junctions in the soleus and extensor digitorum longus muscles of the adult rat. After unilateral section of the sciatic nerve, the proportion of endplates with immunoreactive nerve profiles increased in the denervated muscles even after the axonal material in the distal nerve segments had degenerated. When the nerve was sectioned in the mid-thigh region the proportion of immunoreactive endplates increased with time up to 24-36 h after the nerve section and then eventually declined. The incidence of immunoreactive nerve profiles also increased in the proximal stump. After unilateral sciatic nerve section there was also an increase in the incidence of immunoreactive endplates in the contralateral muscles. The increases in the contralateral muscles could be detected at an earlier time if the nerve was sectioned closer to the spinal cord. The possibility is discussed that the expression of pro-opiomelanocortin peptides in the motor nerves is tonically suppressed in the normal adult but the inhibition is released after section of the nerve. The findings indicate that a signal is transferred from the site of injury in the transected nerves to the motor nerves of the contralateral limb via a transneuronal mechanism through the spinal cord.

Presence of endothelin-1 in porcine spinal cord: isolation and sequence determination.

We investigated the molecular forms of endothelin (ET) related peptides in porcine spinal cord by high performance liquid chromatography coupled with radioimmunoassays using three antisera raised against ET-1 and C-terminal fragments of ET-1 and big ET-1. ET-1 and its oxidized form were isolated as major immunoreactive peptides and sequenced. Furthermore, immunoreactivities like ET-3 and big ET-1(22-39) (contents: less than 8% and less than 1% of ET-1, respectively) were detected based on their chromatographic retention times and characteristics of immunoreactivity to the antisera. Big ET-1 was only scarcely detected. Immunohistochemical study showed the presence of ET-1-like immunoreactivity in motoneurons, dorsal horn neurons and dot- and fiber-like structures in the dorsal horn of lumbar spinal cord. These results indicate that ET-1 is present not only in endothelial cells but also in spinal cord, and that big ET-1 is converted into ET-1 in spinal cord by specific processing between Trp21-Val22. The data also indicate that ET-1 may act as a neuropeptide in the central nervous system.

Distribution of calcitonin gene-related peptide-like immunoreactivity in the medulla oblongata of the cat, in relation to choline acetyltransferase-immunoreactive motoneurones and substance P-immunoreactive fibres.

The immunohistochemical distribution of calcitonin gene-related peptide (CGRP)-like immunoreactivity (ir) in the cat medulla oblongata was examined using an antiserum to rat alpha-CGRP. Comparative distributions of substance P (SP)-like and choline acetyltransferase (ChAT)-like ir were also studied on sections adjacent to those stained for CGRP, and on sections simultaneously stained for CGRP by double staining techniques. The vast majority of ChAT-ir motoneurones in somatomotor or branchiomotor cranial nuclei (of VI, VII and XII nerves) and their accessory nuclei also displayed a coarsely granular CGRP-ir, shown by electron microscopic examination to represent immunoreactive Golgi bodies. The nucleus ambiguus (IX and X nerves), a mixed branchiomotor and visceromotor nucleus, showed CGRP-ir in a lower proportion of its motoneurones, whereas the purely viseromotor dorsal motor vagal nucleus (X nerve) contained no CGRP-ir cells. A few CGRP-ir but ChAT-negative cells were seen in the ventromedial reticular formation, lateral cuneate nucleus, infratrigeminal nucleus and nucleus of the solitary tract. Coarse, often varicose CGRP-ir fibres were most prominent in the X and IX cranial nerve rootlets, the spinal tract of the V nerve and the solitary tract, and also in the V spinal nucleus and nucleus of the solitary tract. Many of these also appeared to contain SP-ir. The central patterns of CGRP and SP-ir fibres thus reflect the previously reported coexistence of these peptides in sensory afferent cells of the trigeminal and nodose ganglia. These results are consistent with a role for CGRP as a transmitter or modulator in efferents to striated muscle, sensory afferents and intrinsic neurones in the cat brain stem.

Glial and neuronal forms of the voltage-dependent sodium channel: characteristics and cell-type distribution.

Two functionally different forms of the voltage-dependent sodium channel were observed in glia and in neurons of the mammalian nervous system. Both forms had identical conductance and tetrodotoxin sensitivity and displayed steady-state inactivation, a strongly voltage-dependent rate of activation, and a faster but weakly voltage-sensitive rate of inactivation. However, the glial form had significantly slower kinetics and a more negative voltage dependence, suggesting that it was functionally specialized for glia. This form was found in most glial types studied, while the neuronal form was observed in retinal ganglion cells, cortical motor neurons, and O2A glial progenitor cells. Both forms occurred in type-2 astrocytes. The presence of the glial form correlated with the RAN-2 surface antigen.

Cranial motor neurons contain either galanin- or calcitonin gene-related peptidelike immunoreactivity.

The demonstration of coexistence of a peptide or peptides in neurons that produce a small molecule neurotransmitter has become increasingly frequent. The calcitonin gene-related peptide (CGRP) is known to be colocalized in the cholinergic neurons of both cranial and spinal motor nuclei. The present study demonstrates that all somatic motor cranial nerve nuclei contain CGRP- and galaninlike immunoreactivity. The perikaryal content of both peptides is increased by colchicine pretreatment and by transecting axons arising from the perikarya, and both peptides are found in nerve fibers innervating striated musculature. CGRP- and galaninlike immunoreactivity appear to be present in different populations of neurons. In contrast to CGRP, galaninlike immunoreactivity was not detected in spinal motor neurons. These observations suggest that galanin and CGRP participate in the process of synaptic transmission at the neuromuscular junction of cranial motor neurons.

[Spinal muscular atrophy: disappearance of RNA fluorescence of degenerating motor neurons. An acridine orange study]. / Atrophie spinale musculaire: l'évanouissement de la fluorescence à l'ARN des neurones moteurs en dégénerescence: une étude à l'acridine-orange.

The histochemical distribution of nucleic acids has been studied in degenerating motor neurons of 9 children who died with spinal muscular atrophy, using the fluorochrome acridine orange. Ribonucleic acid (RNA) fluorescence disappeared abruptly from involved motor neurons without chromatolysis, attenuation of intensity, or other intermediate transitions that follow axotomy or hypoxic insults. We found a nearly identical pattern in 3 adults with amyotrophic lateral sclerosis. The findings in older subjects are complicated, however, by the presence of cytoplasmic lipofuscin. The autofluorescence of this pigment is inhibited by acridine orange. Our results support the hypothesis that spinal muscular atrophy is a disturbance of the genetically coded mechanism that arrests the programmed physiological death of surplus motor neuroblasts after a certain time in embryonic life, so that the normal lethal developmental process becomes pathological by persisting postnatally. A failure of RNA transcription seems to be primary and results in a failure of synthesis of neurotransmitters, of enzymes, and of cytoplasmic proteins. Consequent inanition leads to cell death.

Immunohistochemical evidence for convergence of GABA-containing and glycine-containing axon terminals on single spinal motoneurons of the rat.

GABA- and glycine-containing neuronal elements were visualized in the rat spinal cord by immunohistochemistry employing antisera raised against conjugated GABA and glycine. GABA- and glycine-like immunoreactivities were localized in axon terminals and neuronal perikarya. GABA- and glycine-like immunoreactive terminals were in synaptic contact with perikarya and dendrites of most spinal neurons. Single spinal motoneurons appeared to be in contact with GABA- or glycine-like immunoreactive terminals. These findings suggest that these neurons are regulated postsynaptically by both GABA and glycine.

Motor neurons contain agrin-like molecules.

Molecules antigenically similar to agrin, a protein extracted from the electric organ of Torpedo californica, are highly concentrated in the synaptic basal lamina of neuromuscular junctions in vertebrate skeletal muscle. On the basis of several lines of evidence it has been proposed that agrin-like molecules mediate the nerve-induced formation of acetylcholine receptor (AChR) and acetylcholinesterase (AChE) aggregates on the surface of muscle fibers at developing and regenerating neuromuscular junctions and that they help maintain these postsynaptic specializations in the adult. Here we show that anti-agrin monoclonal antibodies selectively stain the cell bodies of motor neurons in embryos and adults, and that the stain is concentrated in the Golgi apparatus. We also present evidence that motor neurons in both embryos and adults contain molecules that cause the formation of AChR and AChE aggregates on cultured myotubes and that these AChR/AChE-aggregating molecules are antigenically similar to agrin. These findings are consistent with the hypothesis that agrin-like molecules are synthesized by motor neurons, and are released from their axon terminals to become incorporated into the synaptic basal lamina where they direct the formation of synapses during development and regeneration.

Ubiquitin deposits in anterior horn cells in motor neurone disease.

A polyclonal antiserum to ubiquitin, a low molecular weight protein involved in the ATP-dependent removal of abnormal cytoplasmic proteins, has been used to stain spinal cord from 10 cases of motor neurone disease and from 12 control spinal cords. All 10 cases of motor neurone disease exhibited antiubiquitin-immunoreactive deposits in a proportion of the surviving anterior horn cells but these deposits were not seen in any of the 12 controls. These ubiquitin deposits do not correspond to previously described neuronal inclusions in motor neurone disease. The ubiquitin deposits represent, therefore, a new neuronal inclusion which possibly reflects previously unrecognised degradative events occurring in the vulnerable neurones.

Regional heterogeneity in the distal motor axon: three zones with distinctive intrinsic components.

In this study we examined the distribution of cytoskeletal and other intrinsic axonal elements in motor nerve terminals in vivo. Components of axons were visualized with immunocytochemical staining of frozen longitudinal sections of muscle. Using these methods we compared the distribution of neurofilaments, tubulin, MAP2, actin and synaptic elements in distal regions of axons at and near neuromuscular junctions in rat muscles. Our results show that three discrete regions can be defined based on the anatomy and intrinsic components of distal axons. The preterminal axon, extending from its exit from the intramuscular nerve toward the neuromuscular junction, has a cytoskeletal composition similar to the more proximal axon with abundant staining of neurofilaments, tubulin, MAP2 and actin. The terminal arborization, a branched region of the axon extending through the endplate region, contains neurofilaments but little tubulin, actin, MAP2 or synaptic elements. Finally, the synaptic zone, demonstrated with antibodies to the synaptic vesicle protein synaptophysin, contains few cytoskeletal elements. We conclude that there is considerable regional heterogeneity in the composition of distal motor axons. The distribution of neurofilaments, other cytoskeletal elements and synaptic vesicle proteins varies among different discrete zones of terminal motor axons.

Structure and action of buccalin: a modulatory neuropeptide localized to an identified small cardioactive peptide-containing cholinergic motor neuron of Aplysia californica.

A model system that consists of a muscle utilized in biting, the accessory radula closer (ARC), and the two cholinergic motor neurons innervating this muscle, neurons B15 and B16, has been used to study the expression of food-induced arousal in the marine mollusk Aplysia. The ARC muscle receives modulatory input from an extrinsic source, the serotonergic metacerebral cells, which partially accounts for the progressive increase in the strength of biting seen in aroused animals. Another source of modulation may arise from the ARC motor neurons themselves, which synthesize neuropeptides that can potentiate ARC contractions. Neuron B15 synthesizes the two homologous peptides, small cardioactive peptides A and B, whereas neuron B16 synthesizes the structurally unrelated peptide myomodulin. Here we report the purification and sequencing of a neuropeptide termed buccalin and show that it is colocalized with the small cardioactive peptides to neuron B15. Buccalin is also bioactive at the ARC neuromuscular junction but, in contrast to the small cardioactive peptides, when exogenously applied, it decreases rather than increases the size of muscle contractions elicited by firing of the motor neurons. Also unlike the small cardioactive peptides, which exert postsynaptic actions, buccalin seems to act only presynaptically. It has no effect on muscle relaxation rate and decreases motor neuron-elicited excitatory junction potentials in the ARC without affecting contractions produced by direct application of acetylcholine to the muscle. Neuron B15, therefore, appears to contain three modulatory neurotransmitters, two of which may act postsynaptically on the muscle to potentiate the action of the primary neurotransmitter acetylcholine and one of which may act presynaptically on nerve terminals to inhibit acetylcholine release.

Visualisation of CGRP and ChAT-like immunoreactivity in identified trigeminal neurones by combined peroxidase and alkaline phosphatase enzymatic reactions.

We report here a method that allows simultaneous visualisation of two antigens within single neurones. In essence this involves the combined use of horseradish peroxidase and alkaline phosphatase reactions to visualise two markers. Using this method we show that ChAT-and CGRP-like immunoreactivity can be co-localised within single neurones of the V to VII motor nuclei. In the case of the V motor nucleus, we show that each marker can be localised in motoneurones labelled with horseradish peroxidase.